We present a multi-dye ratiometric method for the correction of optical artifacts and unequal illumination encountered by researchers that utilize complex microfluidic systems. Using a novel chemotaxis system that provides cells with passively generated chemoattractant gradients, we demonstrate that the currently utilized method of single-color epi-fluorescence is limited in its ability to characterize gradient formation in the presence of differing channel heights and in the proximity of micron-sized features. As future devices strive to mimic the microtopography of physiological environments, this deficit will become increasingly relevant. The presented multi-color methodology allows for the correction of standard wide-field images and, with the incorporation of laser scanning confocal microscopy, explores three-dimensionally resolved gradient analysis. Using a validated numerical model, we also analyze the potential distortion in gradient formation introduced by device microstructure and the very cells that the system is designed to examine. Our analysis has important implications for the sensitivity of microfabricated systems to cell loading density and channel aspect ratio. In conclusion, the imaging and modeling methodologies introduced in this work will provide complementary information for the rational design and validation of microfluidic devices with cell-sized features.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-12012009-202947 |
| Date | 08 December 2009 |
| Creators | Skandarajah, Arunan |
| Contributors | John P. Wikswo, Christopher J. Janetopoulos |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-12012009-202947/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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